A comprehensive list of genes required for the efficient conjugation of plasmid Rts1 was determined by systematic deletion analysis

Abstract While conjugation-related genes have been identified in many plasmids by genome sequencing, functional analyses have not yet been performed in most cases, and a full set of conjugation genes has been identified for only a few plasmids. Rts1, a prototype IncT plasmid, is a conjugative plasmid that was originally isolated from Proteus vulgaris. Here, we conducted a systematic deletion analysis of Rts1 to fully understand its conjugation system. Through this analysis along with complementation assays, we identified 32 genes that are required for the efficient conjugation of Rts1 from Escherichia coli to E. coli. In addition, the functions of the 28 genes were determined or predicted; 21 were involved in mating-pair formation, three were involved in DNA transfer and replication, including a relaxase gene belonging to the MOBH12 family, one was involved in coupling, and three were involved in transcriptional regulation. Among the functionally well-analysed conjugation systems, most of the 28 genes showed the highest similarity to those of the SXT element, which is an integrative conjugative element of Vibrio cholerae. The Rts1 conjugation gene set included all 23 genes required for the SXT system. Two groups of plasmids with conjugation systems nearly identical or very similar to that of Rts1 were also identified.


Introduction
2][3] Conjugation is mediated by two types of mobile genetic elements (MGEs), conjugative plasmids and integrative conjugative elements (ICEs), the latter of which cannot replicate autonomously but are integrated into host chromosomes in a site-specific manner and can be excised from chromosomes to form circular molecules for conjugation. 4][10][11] Thus, conjugation genes include a set of genes for the T4SS.
The process of assembling the channel complex and sex pili in conjugation is called mating-pair formation (Mpf). 5,12,13e origin of transfer (oriT) is recognized and cleaved by a relaxase.Some auxiliary proteins are also involved in DNA processing; they form a relaxosome along with the relaxase and transferable DNA molecules. 12This process is called DNA transfer and replication (Dtr).The relaxosome and the channel complex are connected by a type IV coupling protein (T4CP).4][15] With an increasing amount of sequence information on conjugative elements from a wide range of bacterial species, gene sets possibly responsible for conjugation functions have been identified based on sequence homologies.However, functional analyses have not yet been performed for most cases.][18][19] Rts1 is a self-transmissible kanamycin (Km)-resistance plasmid that is 217,182 bp in length and was originally found in a clinical isolate of Proteus vulgaris. 20Rts1 is a prototype of the T-incompatibility group 21 and expresses pleiotropic 2 Full gene set for the conjugation of plasmid Rts1 thermosensitive phenotypes in autonomous replication, 22 conjugative transfer, 20 host cell growth, 23 and the restriction 13,14 of T-even phages. 24We previously determined the whole-genome sequence of Rts1. 25 Although the host range of Rts1 has not yet been systematically analysed, it has been shown that Rts1 can be transferred by conjugation from P. vulgaris belonging to the family Morganellaceae which was recently created in the order Enterobacterales 26 to Escherichia coli and Salmonella enterica, both of which belong to the family Enterobactericeae in the order Enterobacterales. 20ur previous homology search of 300 open reading frames (ORFs) on the Rts1 genome identified a set of genes that may be related to conjugation, and many of them showed the noticeable sequence similarity to those of the conjugation systems of plasmids F or R27, which are prototypes of IncFI and IncHI1 plasmids, respectively. 25However, the functions of these genes were not analysed.In this study, to fully understand the conjugation system of Rts1 and identify all of the required components, we conducted a systematic plasmidwide deletion analysis of Rts1.

Bacterial strains, plasmids, and media
Escherichia coli BW25113 (delta(araD-araB)567, delta(rhaD-rhaB)568, delta lacZ4787 (::rrnB-3), and hsdR514, rph-1) 27 was used as the host strain of Rts1 at the gene deletion step and the donor strain in the conjugation assay.Escherichia coli HB101 (leuB6, supE44, thi-1, hsdS20, recA13, ara-14, proAB, lacY1, galK2, rpsL20, xyl-5, and ntl-1) was used as the recipient strain in the conjugation assays.The plasmids pKD46, pKD3, and pCP20 were used for gene deletion. 27lasmids used for the complementation assay were selected from a set of pUC18 derivatives that were previously prepared for random shotgun sequencing of Rts1, 25 and pACYC184 was also used as a cloning vector for the complementation assay for three genes (see the complementation assay section for details).The plasmid pSU4628 (CloDF13::TnA ∆ EcoRV) carrying the ampicillin (Ap)-resistance gene was used for the mobilization assay. 28All strains were cultivated in lysogeny broth (LB) or 2xYT media supplemented with appropriate antibiotics, including Ap at 100 or 200 µg ml −1 , Km at 30 µg ml −1 , streptomycin (Sm) at 200 µg ml −1 , and chloramphenicol (Cm) at 30 µg ml −1 .Rts1 DNA was purified as described previously. 25Routine DNA manipulation and the preparation of genomic and plasmid DNA were carried out by standard methods.DNA sequencing was performed by ABI377 (Applied Biosystems).Escherichia coli strain ER1648 which contains Rts1 25 is available in the Prokaryotes (E.coli) program in the National BioResource Project (https://nbrp.jp/en/resource/prokaryote-en/).

Deletion of genes or gene blocks from the Rts1 genome
Deletion of a gene or a block of genes was introduced into the Rts1 genome by the one-step gene inactivation method, 27 in which the lambda Red recombinase from pKD46 was utilized to facilitate the recombination of linear PCR products into the plasmid genome.pKD3 was used as a template for PCR, and the PCR products were introduced into cells by electroporation using GenePulsar II (Bio-Rad).The PCR products contained the Cm-resistance gene with 50-70 bp tails identical to the sequences that flank the region targeted for deletion.In addition, FLP recognition target sites were introduced on both sides of the Cm-resistance gene to eliminate the resistance gene by the FLP recombinase provided from pCP20.Thus, the gene (or gene block)-deletion mutants that were constructed were resistance marker gene-free and principally avoided polar effects introduced by gene insertion. 27The primers used in this step are listed in Supplementary Table S1.Each deletion was confirmed by the product size of PCR using a pair of outside primers that were used for deletion.

Conjugation and mobilization assays
Escherichia coli BW25113 carrying Rts1 or its mutants (Km-resistant) and HB101 (Sm-resistant) were used as the donor and recipient, respectively.The E. coli cells were cultivated overnight in a 2xYT medium containing appropriate antibiotics at 37°C.Donor and recipient cells were transferred to antibiotic-free fresh 2xYT medium with 100-and 10-fold dilutions, respectively, and the cells were incubated for 3 h at 30°C without shaking.Then, 10 µl of donor cells and 100 µl of recipient cells were mixed with 900 µl of antibioticfree 2xYT medium, incubated for 90 min at 30°C without shaking, and spread on LB plates containing Km and Sm with appropriate dilutions.The transfer frequency of Rts1 (or its mutants) was calculated as the number of transconjugants per donor cell.For the mobilization assay using pSU4628, pSU4628 (encoding the Ap resistance gene) was introduced into BW25113 cells carrying Rts1 or its mutants, which then served as donor cells.The donor and recipient cells were cultivated, mixed, incubated as described above, and spread on LB plates containing Ap and Sm.The transfer frequency of pSU4628 was calculated as the number of pSU4628 transconjugants per donor cell.

Complementation assay
Plasmid clones used for the complementation assay were selected from the pUC18-based random shotgun library of Rts1 used for genome sequence determination 25 based on the end sequences of each insert so that the expression of inserted genes was under the control of the pUC18-encoded lac promoter.pACYC184 served as a cloning vector for three ORFs, Orf191, Orf223, and Orf224, as the pUC18-based clone carrying Orf191 caused adverse effects in terms of host cell growth, and appropriate pUC18-based clones carrying Orf223 and Orf224 were not found in the library.For the cloning of these ORFs into pACYC184, each PCR-amplified DNA fragment was inserted into the BamHI site of pACYC184.The PCR primers used are listed in Supplementary Table S2.
For the complementation assay, these plasmid clones were introduced into donor cells containing a relevant Rts1 mutant by electroporation, and the conjugation assay was performed as described above, except that Ap-or Cm-containing 2xYT medium was used for the cultivation of donor cells carrying pUC18-and pACYC184-derived plasmids, respectively.To avoid overexpression of the inserted genes, isopropyl β-Dthiogalactopyranoside (IPTG) was not added to the culture media during the conjugation assay.The introduction of the empty pUC18 or pACYC184 vector did not alter the conjugation efficiency of Rts1.

Homology search and replicon typing
For sequence homology and domain searches of the ORFs of Rts1, BLASTP (https://www.ncbi.nlm.nih.gov/blast.cgi/) and InterPro (https://www.ebi.ac.uk/interpro/) were used, respectively.To identify conjugation systems similar to that of Rts1, the NCBI database was searched by TBLASTN ver 2.14.1 using the Orf201 and Orf180 protein sequences as queries.GenomeMatcher v3.06 29 was used for the sequence comparison of Rts1 with other plasmids and visualization of the results.PlasmidFinder v2.0.1 30 was used for replicon typing.

Overview and re-evaluation of conjugationrelated genes in Rts1
We re-performed a homology search of all predicted 300 ORFs of Rts1 (Fig. 1) against the genes of functionally wellcharacterized conjugation elements and found homologs not only in plasmids F and R27 but also SXT, a 100-kb multidrug resistance-encoding ICE originally discovered in V. cholerae O139 31,32 whose conjugation system has also been functionally characterized 16,18,19,31,33,34 (Table 1, see Supplementary Table S3 for the revised annotation data of SXT).Of the conjugation systems of the three elements, the ORFs of Rts1 showed the highest similarity to those of SXT.Consistent with this result, the conjugation systems of Rts1 and SXT belong to the MOB H12 family, whereas those of R27 and F belong to the MOB H11 and MOB F12 families, respectively. 35he MPF types of Rts1, SXT and R27 were F type (MFP F ). 36 The gene organization was also relatively well conserved between Rts1 and SXT (Fig. 2).We found that 19 ORFs of Rts1 (Orf196, Orf201, Orf202, Orf204, Orf207-Orf211, Orf213, Orf215-Orf218, Orf240-Orf242, Orf244, and Orf246) exhibited noticeable amino acid sequence similarity to the gene products required for the conjugation of SXT 19,31,33 (Table 1).Although Orf185 and Orf252 did not show noticeable homology to any gene products of SXT, the former showed noticeable homology to TrhP of R27, and the latter contained the MobI domain, suggesting its role in DNA transfer.Two ORFs (Orf224 and Orf248) exhibited noticeable homology to s066 and s082 of SXT, respectively, but these gene products were reported as not required for the conjugation of SXT.19 Orf180 and Orf206 also exhibited sequence similarities to TrbE of F and R0135 of R27, respectively, but TrbE was not required for the conjugation of F, 37 and the function of R0135 is unknown.18,34 Thus, in this analysis, 21 ORFs (the 19 ORFs plus Orf185 and Orf252; indicated by asterisks in Table 1) were identified as potentially related to the conjugation of Rts1. The genes were located in a 60.8-kb region (the region from Orf185 to Orf252 in Fig. 1).The 455-bp region containing oriT, which we experimentally identified in our previous study, 25 was located between orf251 and orf252.

Identification of the conjugation-related genes in Rts1 by systematic deletion analysis
To experimentally determine the necessity of the 21 genes for conjugation and identify additional genes that could be required for the conjugation of Rts1, we performed a systematic deletion analysis of Rts1 by constructing a series of deletion mutants in which a single gene or a block of genes was deleted in E. coli.Finally, we constructed a total of 85 mutants (Fig. 1, Supplementary Table S1), with the largest deletion being a deletion of the 105.4-kb segment encoding orf7-orf158.All Rts1 genes, except for the repA gene encoding a replication initiation protein, were deleted in the mutants.These mutants were then to a conjugation assay using E. coli as the donor and recipient.Note that all mutants were marker-free (see Section 2); thus, it was not necessary to consider the polar effect due to marker gene insertion.
As summarized in Tables 1 and Supplementary Table S1, deletion of 32 genes resulted in significant changes in the conjugation efficiency.The conjugation ability of Rts1 was abolished or severely impaired by the deletion of the abovementioned 21 genes, except for orf218, confirming that these genes were required for conjugation.The orf218 deletion mutant also showed reduced conjugation efficiency, similar to the deletion mutant of the Orf218 homolog in SXT (S063). 19The 11 newly identified genes could be divided into three groups according to the transfer phenotypes of their deletion mutants.Deletion of six genes (orf180, orf186, orf206, orf239, orf247, and orf248) abolished or severely impaired the conjugation ability of Rts1.Of their gene products, two contained domains or motifs indicative of their functions.Orf206 showed homology to thiol oxidoreductase proteins with a thioredoxin fold and the 121Cys-X-X-124Cys motif that can modulate disulfide bonds. 38Orf248 contained the SLT domain of lytic transglycosylases.Although a homolog of transglycosylase was also present in SXT, it was not required for the conjugation of SXT. 19As mentioned above, the Orf180 homolog of F (TrbE) was not required for the conjugation of F, 37 in contrast to Orf180, which was essential for the E. coli-to-E.coli conjugation of Rts1.No significant homologies were found in the remaining three ORFs (Orf186, Orf239, and Orf247).
In contrast to these six genes, deletion of four ORFs (orf191, orf192, orf223, and orf224) provoked a moderate impairment of conjugation efficiency, ranging from a 1/20 to 1/100 reduction compared with the wild type, similar to the orf218-deletion mutant mentioned above (Table 1), indicating that these four ORFs and orf218 are dispensable but required for the efficient E. coli-to-E.coli conjugation of Rts1.Orf223 and Orf224 contained RecT and phage_rel_nuc domains, respectively, and Orf224 showed notable homology to S066 of SXT, which was not required for the conjugation of SXT. 19No noticeable sequence homology was found for Orf191 and Orf192.
The deletion mutant of the remaining ORF (orf249) exhibited a completely different phenotype.This mutant showed remarkably improved conjugation efficiency, a 400-fold improvement compared with the wild type; no significant homology was found for this ORF.

Complementation analysis
To confirm that the products of the 32 genes identified by deletion analysis were actually involved in conjugation, we performed a systematic complementation analysis.From this analysis, we excluded 14 ORFs (orf201, orf202, orf207-211, orf213, orf216, orf217, orf240-242, and orf244) because their essentiality was apparent from their sequence homologies to known essential genes in the other conjugation systems.In this analysis, we utilized plasmid clones in the pUC18-based Rts1 genomic library, which was used for whole genome random shotgun sequencing and whose end sequences were known. 25Specifically, we selected plasmid clones containing an entire target gene (Supplementary Fig. S1) and introduced them into E. coli harbouring the corresponding deletion mutant of Rts1.Complementing genes were expressed by the lacZ promoter on pUC18 without induction by IPTG.For Table 1.Continued the complementation of orf191, and orf224, we used the low copy number plasmid pACYC184 as a vector plasmid (Supplementary Fig. S1 and Table S2) because appropriate pUC18 derivatives were not found (orf223 and orf224) or the introduction of plasmid clone resulted in the inhibition of host cell growth (orf191).In the case of orf223 and orf224, a pACYC184 derivative carrying both genes was used.
Of the 18 deletion mutants analysed (Table 1), the conjugation efficiencies of 17 mutants that showed diminished or highly impaired conjugation were improved by introducing complementing plasmid clones, although the efficiencies of several mutants were not restored to the wild-type level.Interestingly, complementation of orf246-and orf247deletion mutants resulted in a remarkable improvement in conjugation efficiency, with 1,489-and 97-fold increases compared with wild type, respectively (Table 1).In contrast, complementation of the orf249-deletion mutant, which showed an increased conjugation ability, resulted in a 1/40fold reduction in conjugation efficiency (Table 1).These results suggested that orf246 and orf247 positively regulate the gene expression of the Rts1 conjugation system and orf249 negatively regulates this expression.

Mobilization assay of plasmid pSU4628 in Rts1 mutants
To elucidate the roles of the 32 genes involved in the conjugation process, we conducted a systematic mobilization assay of each deletion mutant using the plasmid pSU4628, an Ap-resistant derivative of the plasmid CloDF13. 28pSU4628 is not self-transmissible but is mobilizable if Mpf gene products are provided by a co-resident helper conjugative element, as pSU4628 encodes its own Dtr proteins and T4CP. 28In this analysis, two mutants of presumed positive regulator-encoding genes (orf246 and orf247) were unable to mobilize pSU4628, as expected (Table 1).Of the remaining conjugation-deficit mutants, 17 were unable to mobilize pSU4628 (Table 1), indicating that the 17 genes deleted in these mutants are required for Mpf.In addition, four genes (orf185, orf191, orf206, and orf248) appeared to impact Mpf because their deletion mutants showed very low mobilization.In contrast, eight conjugation-deficit mutants (orf192, orf201, orf202, orf204, orf218, orf223, orf224 and orf252-deletion mutants) were able to mobilize pSU4628 as efficiently as wild-type Rts1 (Table 1).This result indicates that these eight genes are not involved in Mpf.The deletion mutant of orf249, which encodes a presumed negative regulator, showed a markedly increased ability to mobilize pSU4628, as expected.

Plasmids with a conjugation system highly homologous to the Rts1 system
By searching genes similar to Orf201 (relaxase) in Rts1, we identified 14 plasmids that encode conjugation systems almost identical or very similar to that of Rts1 (Table 2).Five plasmids from Providencia, Escherichia, and Proteus species contained a region almost identical to the 64.6-kbregion of Rts1 where conjugation-related genes are located (Figs. 3A and 4).Their rep genes were also nearly identical (>99.8%nucleotide sequence identity) to that of Rts1 and belonged to the IncT group.However, these plasmids were much smaller than Rts1, and various sizes of regions between the rep gene and the conjugation-related region where the Km-resistance gene is located in Rts1 were missing (Fig. 3A).Nine plasmids from Klebsiella and Citrobacter species also contained a region similar to the conjugation-related region of Rts1 (Fig. 3B, see Supplementary Fig. S2 for the comparison of all members in this group), but the amino acid sequence identities of each gene product were 23-92% of the counterparts of Rts1 (Fig. 4).In addition, only the conjugation-related region showed sequence similarities to Rts1 (Fig. 3B, see also Supplementary Fig. S2), and the Inc type was 'repA(pKOX)', as defined by PlasmidFinder.Although many plasmids from various species were found to encode an Orf201 homolog more similar to that of SXT, their amino acid sequence identities to Orf201 were less than 51%.Of note, the homologs of Orf180, which was not present in SXT but essential for the E. coli-to-E.coli conjugation of Rts1, were present only in the abovementioned two groups of plasmids (Table 2).

Discussion
Through a systematic plasmid-wide deletion analysis followed by complementation analyses, we identified a full set of genes required for the efficient conjugation of Rts1 from E. coli to E. coli, which included 32 genes (Table 1).Of the 32 genes, 23 were essential (conjugation-undetectable or severely impaired for their deletion mutants), and nine were not essential but required for efficient or controlled conjugation (Table 1).Based on the results of mobilization assays along with the phenotypes of deletion mutants and the results of the homology search, genes involved in Mpf and Dtr, the gene encoding the T4CP protein, and the genes involved in regulating the expression of the Rts1 conjugation system were identified, as discussed below.Plasmids with a conjugation system almost identical or very similar to that of Rts1 were  also identified search (Table 2, Figs. 3 and 4, and Supplementary S2).

Mpf genes
The results of mobilization assays suggested that 21 genes are involved in Mpf, of which three (orf186, orf191, and orf239) showed no noticeable homologies to the genes in SXT, R27, and F (Table 1).Of the 21 genes, the essential requirement of orf180, which encodes a small protein of 45 amino acids and is conserved in close relatives of Rts1, is interesting because its homolog in plasmid F was not required for conjugation (Table 2 and Fig. 3).The presence of orf185, which appears to encode a LepB-like type I signal peptidase (peptidase S26 superfamily; pfam PF00717) with predicted catalytic residues (Ser56 and Lys101) and domains B-E conserved among the type I signal peptidase family, was also interesting. 39Rts1 encodes another peptidase, Orf215, which is also required for Mpf and belongs to the TraF protein family with the active residues Lys67 and Asp130. 40,41Thus, two peptidases are required for the E. coli-to-E.coli conjugation of Rts1.To the best of our knowledge, Rts1 is the first conjugative element found to encode more than one conjugation-related protease.In plasmid RP4, TraF processes TrbC, a major component of sex pili, at the C-terminus for its maturation and cyclization, and two host-encoded peptidases, LepB and an unknown peptidase, are necessary for the maturation of TrbC. 42,43In Rts1, the pilin protein (Orf196) or some other proteins may require processing by Orf185, but the precise function and substrates of this LepB homolog remain to be elucidated.
Another interesting Mpf gene identified is orf248, which is located between the regulator genes and encodes a member of the soluble lytic transglycosylase (SLT) family with three conserved motifs and a presumed catalytic residue, Glu55. 44LTs are widely distributed among type III secretion systems and T4SSs and are thought to be involved in rearrangement of the peptidoglycan layer to form the transport machinery required for penetrating the cell wall. 44However, an Orf248 homolog in SXT was not required for conjugation. 19R27 also encodes an SLT family protein, but its function has not yet been analysed. 18,34he presence of Orf206 was also interesting, as it contains a thioredoxin fold with a catalytic CXXC motif, which implicates it as a thiol disulfide oxidoreductase that catalyses intrachain disulfide bond formation in extracytoplasmic proteins.6][47][48] The contribution of these thioredoxin-like proteins to conjugation has also been reported in F, R27, and SXT. 19,45,46Although the deletion of the genes for these proteins in F (trbB) and R27 (dsbC) reduced their conjugation efficiency (10-fold and 10,000-fold reduction, respectively), the reduction was observed only in dsbC-null host strains, suggesting the redundancy of hostand plasmid-encoded thioredoxins.In contrast, deletion of orf206 resulted in a >100-fold decrease in the conjugation efficiency of Rts1, even in the dsbC-positive host strain, suggesting that the function of Orf206 is unique and specific to Rts1.A host-encoded dsbC-independent phenotype was also observed in SXT, where the deletion of a dsbC-homolog

Dtr and T4CP genes
Among the eight genes other than Mpf-related genes and regulatory genes, four (orf201, orf202, orf204, and orf252) were found to be essential for the E. coli-to-E.coli conjugation of Rts1 (Table 1).Of these, from the results of the homology search, orf201 and orf202 apparently encode the relaxase and  3. Dot plot matrix analysis of the plasmids with conjugation-related regions nearly identical or similar to that of Rts1.(A) Genomic comparison of Rts1 with six plasmids that possessed a conjugation-related region nearly identical to that of Rts1.(B) Genomic comparison of Rts1 with three plasmids with conjugation-related regions similar to that of Rts1.See Supplementary Fig. S2 for the results of dot plot matrix analysis of all nine plasmids with conjugation-related regions similar to that of Rts1.In both panels, conjugation-related regions are indicated by light grey shading.T4CP of Rts1, respectively.Orf204 contains a domain of unknown function (DUF4400), and Orf252 contains the MobI domain, which has been shown to be responsible for the recognition of the oriT locus in IncC plasmids. 49Conjugative elements usually encode a couple of auxiliary proteins involved in Dtr. 12 For instance, TraY and TraM in IncF plasmids and TraH, TraJ, and TraK in IncP plasmids are auxiliary proteins of Dtr that determine the oriT specificity or unwinding of the specific locus of the DNA helix to initiate DNA transfer. 12According to these well-characterized plasmids, orf204 and orf252 presumably play auxiliary functions in DNA metabolism during the conjugation process.Consistent with this, SXT and R27 also contain two auxiliary proteins of Dtr that are essential for their conjugation, 19,33,34 and Orf204 showed noticeable homology to one of the auxiliary proteins (TraJ) of SXT and R27 (Table 1).Although Orf252 did not show significant sequence similarities to the protein named MobI in SXT and R27, the genes encoding these proteins are directly located next to the oriT locus in SXT and R27, as seen for orf252 of Rts1 (see Fig. 2 for the SXT data).

Regulatory genes
The phenotypes of deletion mutants of three ORFs (Orf246, Orf247, and Orf249) suggested that the E. coli-to-E.coli conjugation of Rts1 is negatively regulated by Orf249 and positively regulated by Orf246 and Orf247.Although no significant sequence homology was detected between Orf249 and SetR of SXT, which was experimentally identified as a repressor, 31 Orf249 appears to be a functional homolog of SetR.Furthermore, Orf246 showed homology to SetC, one of the two positive regulators of SXT, and showed homology to FlhC, one of the master regulators of flagella biosynthesis in E. coli and Salmonella.FlhC forms a hexameric complex with FlhD to work as a transcriptional activator of the class 2 operon, thereby the expression of flagellar genes. 50,51he flhC is located just downstream of flhD in E. coli.Rts1, orf246 is also located just downstream of orf247.In SXT, setD, a homolog of flhD, is also located next to setC (Fig. 2), and SetC and SetD were experimentally shown to work as transcriptional activators by binding to several promoters of conjugation genes. 52][55] Considering these observations, Orf246 and Orf247 likely form a heteromeric complex to work as activators.

Other genes required for the efficient E. colito-E. coli conjugation of Rts1
The roles of the remaining four gene products (Orf192, Orf218, Orf223, and Orf224) are currently unknown, but the conjugation efficiencies of their deletion mutants were 1/10-1/100 of that of wild-type Rts1 (Table 1).Of these, Orf223 and Orf224 contained the RecT and phage_rel_nuc domains, respectively.Interestingly, S065 and S066 of SXT also contained these domains, and the latter showed noticeable homology to Orf224.These SXT genes were named bet and exo based on the homologies to the bet and exo genes of lambda phage and experimentally shown to have RecA-independent homologous recombination activity similar to the lambda system. 56,57However, in contrast to Rts1, these genes were not required for the conjugation of SXT. 19Orf218 also showed sequence similarity to S063 of SXT, and the s063-deletion mutant and the orf218-deletion mutant both showed a reduction in conjugation efficiency 19 (Table 1), but the functions of S063 are also currently unknown.The function of orf192 also remains to be investigated, as no significant sequence homology to known functionally characterized genes was found.
described in Section 3.1 are indicated by asterisks.b Efficiency relative to that of the wild type Rts1.c NT, not tested.d Orf216 contains the TrbC_F type domain and the TraW domain.e Not required for conjugation.f Unknown for their involvement in conjugation.

Figure 2 .
Figure 2. Comparison of the conjugation-related regions of Rts1 and SXT.The gene organizations of the conjugation-related regions of Rts1 with SXT and amino acid sequence homologies between genes are shown.Note that traI is located next to mobI in the SXT element.

Table 1 .
The 32 genes required for the efficient conjugation of Rts1 from Figure 1.The gene organization of Rts1 and deletion mutants constructed in this study.The numbers under each line indicate the deleted ORF(s).Conjugation-related genes identified in this study are shown in red, and those for plasmid replication and maintenance are shown in yellow.The Km-resistance gene carried by Tn2680 is indicated in blue.

Table 2 .
Plasmids encoding proteins highly homologous to Orf201 and Orf180 of Rts1.
(s054) abolished its conjugation in a dsbC-positive E. coli Interestingly, S054 is a homolog of Orf212 in Rts1, but Orf212 is not required for the conjugation of Rts1 from E. coli to E. coli.Although the reason for this difference is unknown, some effect of orf212 deletion may be observed in the orf206-negative background.